Tissue transglutaminase (tTG) is a novel, dual function protein that is both a calcium-dependent transamidating enzyme and a signal transducing GTP-binding protein (Galpha h). As a transamidating enzyme, tTG catalyzes the formation of isopeptide bonds between specific substrate proteins to produce insoluble polymeric structures. A defining characteristic of Alzheimer's disease brain is the presence of intracellular (neurofibrillary tangles [NFTs]) and extracellular (senile plaques) filamentous proteinaceous aggregates that are highly insoluble. Studies from this, and other laboratories, have demonstrated that tau, the major protein of the NFTs, and Abeta (1-40), a primary peptide of the senile plaques, are both excellent in vitro substrates of tTG. Recent studies from the applicants laboratory have demonstrated that in cerebral cortex, where NFTs and senile plaques are prevalent, but in cerebellum which is virtually devoid of these lesions, tTG levels and TG activity are elevated significantly in Alzheimer's disease brain compared to age-matched controls. In addition, it has been hypothesized that tTG maybe involved in the neurodegeneration of codon reiteration diseases, such as Huntington's disease, by facilitating the formation of insoluble neuronal inclusions. These and other findings indicate that tTG could contribute to the formation of the insoluble, pathological lesions in certain neurodegenerative disorders. The focus of this proposal, which is a competing continuation, is on investigating the direct and indirect in situ regulation of tTG, predominantly by calcium and GTP, and how these processes may be disrupted, especially in conditions associated with Alzheimer's disease. This focus on the modulation of tTG, in situ represents a significant advance compared to the many previous in vitro studies. The applicants comprehensive working hypothesis is that in situ tTG is tightly regulated, and that perturbations of these regulatory processes results in inappropriate increases in the levels and transamidating activity of tTG and this contributes to the neurodegenerative processes of Alzheimer's disease. In this proposal the majority of experiments will be carried out in human neuroblastoma cells, although primary cell cultures of rat cerebral cortical neurons, as well as hippocampal neurons, will also be used in some studies. The goals of this proposal are to test the following hypotheses (1) that GTP and calcium work in concert to regulate tTG activity through direct and indirect mechanisms, (2) that receptor-mediated mobilization of calcium from the endoplasmic reticulum (ER) plays a significant role in modulating the transamidating activity of tTG, (3) that activation of the transamidating activity of tTG results in the modification of tau, and these modifications are associated with specific alterations in the metabolism, function and subcellular distribution of tau, (4) that GTP modulates tTG interactions with specific proteins which direct the localization and determine the function of tTG (i.e., as a transamidating enzyme or signal transducing G protein), and (5) that Abeta and/or Alzheimer's presenilin mutants increase the transamidating activity of tTG by direct and/or indirect mechanisms. These studies will increase our understanding of the regulation of tTG significantly and are likely to provide insight into its putative role in neurodegenerative processes.